potassium (oxido-NNO-azoxy)cyclohexane; cyclohexylhydroxydiazene 1-oxide, potassium salt; [K-HDO]

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 14.10.2004; Experimental start date: 18.10.2004; Experimental termination date: 16.11.2004; Study completion date: 25.01.2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Version / remarks:

adapted 21 st January 2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Terrestrial Ecotoxicology Under Council Directive 91/414/EEC. SANCO/10329/2002

Version / remarks:

rev. 2 final. Working Document (Draft), 17 October 2002.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

EC Number:

613-953-8

Cas Number:

66603-10-9

Molecular formula:

C6H11KN2O2

IUPAC Name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

Test material form:

liquid

Specific details on test material used for the study:

Lot/Batch number: VM 2012
Purity: 30 % (30.41 % analysed) in water
Formulation type: aqueous solution
Appearance: yellowish liquid
Density: 1.12 g/cm³ at 25 °C
Received: 27.09.2004
Expiration date: 28.07.2006
Chemical name: N-Cyclohexyldiazeniumdioxy-potassium
Solubility in water: Soluble
Storage conditions: room temperature (> 15 °C)
Stability: expected to be stable at least for 2 years if stored under the cited storage conditions
Test concentrations: 20 mg/kg, 100 mg/kg soil dry weight (d.w.)

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

Soil samples (100 g d.m.) are taken at intervals of 3 hours, 7, 14 and 28 days after application of the test item. Before the beginning of the test, the optimal glucose concentration was determined

Test substrate

Vehicle:

yes

Remarks:

water

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Biologically active agricultural soil: loamy sand soil.


SOIL REMOVAL, SOIL PREPARATION AND SOIL STORAGE
Corresponding to the OECD Guideline an agriculturally utilized soil was selected. The soil was removed to a depth of 20 cm as mixed samples. Because the soil was wet it was carefully dried at room temperature. Afterwards, the soil was passed through a 2 mm mesh sieve. Subsequently, the soil was stored at a temperature of 4 °C in containers under aerobic conditions in the dark.

TEST PREPARATION
The examination was performed with one soil.
The test item was applied at a test concentration of 20 mg/kg soil d.w. and a fivefold test concentration of 100 mg/kg soil d.w. The short-term respiration was determined on day 0 (after approximately 3 hours) and at intervals of 7, 14, and 28 days after application.

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

(20 ± 2) °C in a climatic room

Moisture:

Water content of soil: approximately 45 % of WHC

Details on test conditions:

TEST SYSTEM
- Testing facility: Biochem Agrar
- Test container (type, material, size): Soil samples were incubated at 20 °C ± 2 °C while stored in new plastic vessels.
- Amount of soil: 1200 g soil d.w. per vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- The incubation of the prepared soil was carried out in new plastic vessels ( 4 L)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): 12.694435960 degrees East, 51.403774567 degrees North
- History of site: fallow ground
- Vegetation cover: fallow ground
- Treatments with pesticides or fertilizers: none
- Accidental contamination: none
- Depth of sampling: The soil was removed to a depth of 20 cm as mixed samples.
- Humus content [%]: 2.51
- Nmin [mg/100 g soil d.w.]: 0.99
- % sand: 50.2
- % silt: 39.1
- % clay: 10.7
- Soil taxonomic classification: loamy sand soil.
- pH (in water): 6.6 before application, 6.4 after 28 days
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weigth): 45 % of WHC
- Cation exchange capacity (mmol/kg): 12.15
- Pretreatment of soil: Because the soil was wet it was carefully dried at room temperature. Afterwards, the soil was passed through a 2 mm mesh sieve.
Subsequently, the soil was stored at a temperature of 4 °C in containers under aerobic conditions in the dark.
- Storage (condition, duration): the soil was stored at a temperature of 4 °C in containers under aerobic conditions in the dark.
- Initial microbial biomass as % of total organic C: 24.37 = 1.67 % compared to Corg.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : pH-value, water content, O2 consumption. Soil samples (100 g soil d.w.) were taken at intervals of 3 hours, 7, 14 and 28 days after application of the test item.

RANGE-FINDING STUDY
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

dinoseb acetate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

28 d

Dose descriptor:

other: O2 consumption- deviation from control

Effect conc.:

1.9 other: - % deviation from control with a test concentration of 100 mg/kg soil dry weight

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: O2 consumption

Remarks on result:

other: No adverse effects on carbon transformation

Effect concentrations 2

Key result

Duration:

28 d

Dose descriptor:

other: O2 consumption deviation - from control

Effect conc.:

0.3 other: 0.3 % deviation from control with a test concentration of 20 mg/kg soil dry weight

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: O2 consumption - deviation from control

Remarks on result:

other: No adverse effects on carbon transformation

Details on results:

See table below

Results with reference substance (positive control):

The reference item dinoseb acetate produced in the soil the expected level of effect (25.4 % inhibition after 28 days).

Reported statistics and error estimates:

Calculation of mean values per treatment, standard deviations and coefficients of variation.

Any other information on results incl. tables

Effects on carbon transformation in soil after treatment with Xyligen LP 15684
Days after application	Control		Xyligen LP 15684 20 mg/kg soil d.w.			Xyligen LP 15684 100 mg/kg soil d.w.
	O2consumption (mg/kg soil d.w./h)	CV (%)	O2consumption (mg/kg soil d.w./h)	CV (%)	Deviation from control (%)	O2consumption (mg/kg soil d.w./h)	CV (%)	Deviation from control (%)
0	7.99	0.87	8.15	0.41	+ 2.0	7.76	0.96	-2.9
7	8.06	0.67	8.06	0.35	0.0	7.83	0.65	-3.0
14	7.78	0.99	7.83	1.45	+ 0.6	7.61	0.49	-2.1
28	7.31	1.75	7.28	0.51	- 0.3	7.17	2.22	-1.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria:
The coefficients of variation in control were maximum 1.75 % and thus fulfilled the demanded range (≤ 15 %). In the most recent test, dated 14.01.04 – 11.02.04, the toxic standard Dinoseb acetate caused a reduction of the O2-consumption of 25.4 % after 28 days and thus demonstrated the sensitivity of the test system.
No short-term and long-term effects (OECD 217) on the carbon transformation in the field soil tested up to a concentration of 100 mg Xyligen LP 15684 per kg soil dry weight.


No short-term and long-term effects (OECD 217) on the carbon transformation in the field soil tested up to a concentration of 100 mg Xyligen LP 15684 (30 % K-HDO in water) per kg soil dry weight.

Executive summary:

Based on the results of this study Xyligen LP 15684 (K-HDO 30 % in water) caused no short-term and long-term effects (OECD 217) on the carbon transformation in the field soil tested up to a concentration of 100 mg Xyligen LP 15684 per kg soil dry weight.